EP3775215A1 - Procédés de modulation de l'activité antisens - Google Patents

Procédés de modulation de l'activité antisens

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Publication number
EP3775215A1
EP3775215A1 EP19782173.9A EP19782173A EP3775215A1 EP 3775215 A1 EP3775215 A1 EP 3775215A1 EP 19782173 A EP19782173 A EP 19782173A EP 3775215 A1 EP3775215 A1 EP 3775215A1
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EP
European Patent Office
Prior art keywords
autophagy
certain embodiments
modified
oligonucleotide
antisense
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP19782173.9A
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German (de)
English (en)
Inventor
Joseph OCHABA
Mariam Aghajan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ionis Pharmaceuticals Inc
Original Assignee
Ionis Pharmaceuticals Inc
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Filing date
Publication date
Application filed by Ionis Pharmaceuticals Inc filed Critical Ionis Pharmaceuticals Inc
Publication of EP3775215A1 publication Critical patent/EP3775215A1/fr
Withdrawn legal-status Critical Current

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/313—Phosphorodithioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
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    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • Autophagy is a process in which cellular components are destroyed by being taken up in vesicles along with a portion of a cell’s cytoplasm and subsequently delivered to lysosomes for degradation and recycling. This process is part of normal cell homeostasis and is involved in many physiologic functions, including immunity, cell survival, development, and differentiation.
  • antisense compounds including antisense oligonucleotides
  • uptake pathways of antisense compounds that result in pharmacological effects are referred to as productive uptake pathways.
  • the present disclosure provides methods of increasing antisense activity by modulating autophagy.
  • the methods provided herein comprise contacting a cell with an antisense compound and contacting a cell with an autophagy modulator.
  • the autophagy modulation is activation of autophagy.
  • the autophagy modulation is blocking the fusion of autophagosomes with lysosomes.
  • the autophagy modulation increases antisense activity by increasing productive uptake of the antisense compound.
  • the antisense activity of the antisense compound is reduction of the level of a target nucleic acid.
  • the antisense activity of the antisense compound is splicing modulation of a target nucleic acid.
  • the antisense activity of the antisense compound is increase of the level of a target nucleic acid.
  • the methods herein comprising autophagy modulation result in an extent of antisense activity that is greater than the extent of antisense activity that occurs when autophagy is not modulated.
  • autophagy modulation that results in increased antisense activity may occur at one or more steps of the autophagy pathway.
  • some autophagy modulators that increase antisense activity increase the rate of autophagosome formation and/or the number of autophagosomes present in the cell at a given time after autophagy modulation.
  • Autophagosome formation involves multiple steps, including autophagosome nucleation, elongation, sequestration of cytoplasmic contents, and completed formation of an intact autophagosome. See, e.g.
  • Antisense compounds in productive uptake pathways may be contained in some autophagosomes and/or may shuttle between autophagosomes and/or other intracellular vesicles.
  • autophagosomes can fuse temporarily or permanently with endosomes.
  • autophagosomes fuse with lysosomes to form autolysosomes.
  • autolysosomes are acidified and their contents are destroyed.
  • autolysosomes break apart and revert to separate autophagosomes and lysosomes.
  • antisense compounds that were headed for degradation in an unproductive uptake pathway may be redirected to a productive uptake pathway.
  • “2’-deoxynucleoside” means a nucleoside comprising 2’-H(H) ribosyl sugar moiety, as found in naturally occurring deoxyribonucleic acids (DNA).
  • a 2’- deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil).
  • 2’-fluoro or“2’-F” means a 2’-F in place of the 2’-OH group of a ribosyl ring of a sugar moiety.
  • “2’-substituted nucleoside” or“2 -modified nucleoside” means a nucleoside comprising a 2’-substituted or 2’-modified sugar moiety.
  • “2’-substituted” or“2 -modified” in reference to a sugar moiety means a sugar moiety comprising at least one 2'-substituent group other than H or OH.
  • antisense activity means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid.
  • antisense activity is a decrease in the amount or expression of a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.
  • antisense activity is an increase in the amount or expression of a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.
  • antisense activity is a modulation of splicing a target nucleic acid compared to target nucleic acid splicing in the absence of the antisense compound.
  • antisense compound means a compound comprising an antisense oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group.
  • antisense oligonucleotide means an oligonucleotide having a nucleobase sequence that is at least partially complementary to a target nucleic acid.
  • “ameliorate” in reference to a method means improvement in at least one symptom of a disease or condition and/or measurable outcome relative to the same symptom or measurable outcome in the absence of or prior to performing the method.
  • amelioration comprises a reduction in severity of a symptom, reduction in frequency of a symptom, delayed onset of the disease or condition, slowing of progression of the disease or condition, or a combination thereof.
  • ATP-competitive inhibitor is a kinase inhibitor that binds to the ATP binding site of one or more kinases.
  • ATP-competitive inhibitor is bound to the ATP binding site of a kinase
  • autophagy activation means a modulation of autophagy in which the perturbation results in an increase of one or more steps or components of autophagy relative to the level of autophagy that occurs in the absence of the autophagy activation.
  • the level of autophagy that occurs in the absence of the autophagy activation is that which is observed under conditions that are otherwise the same except that an autophagy activator is not present.
  • an autophagy activator is a compound or composition that activates autophagy in a cell after contacting the cell.
  • an autophagy activator is a condition that activates autophagy.
  • the condition that activates autophagy is fasting.
  • the condition that activates autophagy is nutrient restriction (e.g., ketogenic diet).
  • autophagosome formation means a process that begins with nucleation of a phagophore and ends with completed formation of an intact autophagosome.
  • autophagy modulation means a perturbation of activity, size, amount, and/or the rate of a step or component in autophagy.
  • Steps in autophagy include phagophore nucleation, elongation, sequestration of cytoplasmic components, autophagosome formation, fusion of autophagosome to endosome, fusion of autophagosome to lysosome, and degradation of the components of the autolysosome.
  • Components in autophagy include proteins and other molecules that contribute to carrying out the steps of autophagy.
  • an“autophagy modulator” is a compound or composition that modulates autophagy in a cell after contacting the cell. Processes that affect autophagy that do not involve contacting a cell with a compound or composition, such as fasting or nutrient restriction, are not autophagy modulators.
  • autophagic vesicle means a double membraned vesicle that is physically associated with LC3-II.
  • basal or“basal level” means the level or amount of a process observed in the absence of a particular stimulus.
  • a basal level of autophagy is the level of autophagy observed in the absence of an autophagy modulator or a stimulus that modulates autophagy, such as fasting.
  • “bicyclic nucleoside” or“BNA” means a nucleoside comprising a bicyclic sugar moiety.
  • “bicyclic sugar” or“bicyclic sugar moiety” means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure.
  • the first ring of the bicyclic sugar moiety is a furanosyl moiety.
  • the bicyclic sugar moiety does not comprise a furanosyl moiety.
  • “cEt” or“constrained ethyl” means a ribosyl bicyclic sugar moiety wherein the second ring of the bicyclic sugar is formed via a bridge connecting the 4’-carbon and the 2’-carbon, wherein the bridge has the formula 4'-CH(CH 3 )-0-2', and wherein the methyl group of the bridge is in the S configuration.
  • cleavable moiety means a bond or group of atoms that is cleaved under physiological conditions, for example, inside a cell, an animal, or a human.
  • “complementary” in reference to an oligonucleotide means that at least 70% of the nucleobases of such oligonucleotide or one or more regions thereof and the nucleobases of another nucleic acid or one or more regions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions.
  • “complementary nucleobases” means nucleobases that are capable of forming hydrogen bonds with one another.
  • Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5 -methyl cytosine ( m C) and guanine (G).
  • Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. As used herein,“fully complementary” or“100% complementary” in reference to
  • oligonucleotides means that such oligonucleotides are complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide.
  • conjugate group means a group of atoms that is directly or indirectly attached to an oligonucleotide.
  • Conjugate groups include a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.
  • conjugate linker means a group of atoms comprising at least one bond that connects a conjugate moiety to an oligonucleotide.
  • conjugate moiety means a group of atoms that is attached to an oligonucleotide via a conjugate linker.
  • oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or intemucleoside linkages that are immediately adjacent to each other.
  • contiguous nucleobases means nucleobases that are immediately adjacent to each other in a sequence.
  • COPB2 coatomer protein complex subunit beta 2.
  • COPB2 is encoded by the COPB2 gene.
  • double-stranded antisense compound means an antisense compound comprising two oligomeric compounds that are complementary to each other and form a duplex, and wherein one of the two said oligomeric compounds comprises an antisense oligonucleotide.
  • expression in reference to a gene or the product of a gene means the amount of a nucleic acid or protein that is encoded from a gene or encoded from a product of a gene.
  • a change in expression such as inhibition of expression, may be either a change to an encoding step itself (e.g., transcription or translation) or a change to the level of the encoded nucleic acid or protein, independent of any effects on the encoding step.
  • “fully modified” in reference to a modified oligonucleotide means a modified oligonucleotide in which each sugar moiety is modified.
  • “Uniformly modified” in reference to a modified oligonucleotide means a fully modified oligonucleotide in which each sugar moiety is the same.
  • the nucleosides of a uniformly modified oligonucleotide can each have a 2’-MOE modification but different nucleobase modifications, and the intemucleoside linkages may be different.
  • “gapmer” means an antisense oligonucleotide comprising an internal“gap” region having a plurality of nucleosides that support RNase H cleavage positioned between external“wing” regions having one or more nucleosides, wherein the nucleosides comprising the internal gap region are chemically distinct from the terminal wing nucleosides of the external wing regions.
  • hybridization means the pairing or annealing of complementary oligonucleotides and/or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
  • inhibiting refers to a partial or complete reduction.
  • inhibiting the expression of a target nucleic acid means a partial or complete reduction of expression of the nucleic acid, e.g. , a reduction in the amount of protein produced from the target nucleic acid, and does not necessarily indicate a total elimination of the protein or target nucleic acid.
  • Selective inhibition is inhibition that occurs to a significantly greater extent for at least one identified target than for at least one other identified target.
  • a kinase inhibitor that is selective for kinase X over kinase Y inhibits kinase X to a significantly greater extent than kinase Y.
  • intemucleoside linkage means a group or bond that forms a covalent linkage between adjacent nucleosides in an oligonucleotide.
  • modified intemucleoside linkage means any intemucleoside linkage other than a naturally occurring, phosphate intemucleoside linkage. Non-phosphate linkages are referred to herein as modified intemucleoside linkages.
  • Phosphorothioate linkage means a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced with a sulfur atom.
  • a phosphorothioate intemucleoside linkage is a modified
  • Modified intemucleoside linkages include linkages that comprise abasic nucleosides.
  • LC3 means microtubule-associated protein lA/lB-light chain 3.
  • the LC3 protein is encoded by the MAP1LC3A gene.
  • LC3-I is a cytosolic form of LC3.
  • LC3-II is an LC-3- phosphatidylethanolamine conj ugate .
  • linker-nucleoside means a nucleoside that links, either directly or indirectly, an oligonucleotide to a conjugate moiety. Linker-nucleosides are located within the conjugate linker of an oligomeric compound. Linker-nucleosides are not considered part of the oligonucleotide portion of an oligomeric compound even if they are contiguous with the oligonucleotide.
  • “linked nucleosides” are nucleosides that are connected in a continuous sequence (i.e. no additional nucleosides are present between those that are linked).
  • mismatch or“non-complementary” means a nucleobase of a first oligonucleotide that is not complementary with the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligomeric compound are aligned.
  • “MOE” means methoxyethyl.”2’-MOE” means a 2’-OCH 2 CH 2 0CH 3 group in place of the 2’-OH group of a ribosyl ring of a sugar moiety.
  • “motif’ means the pattern of unmodified and/or modified sugar moieties, nucleobases, and/or intemucleoside linkages, in an oligonucleotide.
  • mTor means mammalian target of rapamycin. mTor is a protein kinase that is encoded by the MTOR gene.
  • non-bicyclic modified sugar or“non-bicyclic modified sugar moiety” means a modified sugar moiety that comprises a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.
  • nucleic acid delivery vehicle means a cationic polymer, liposome, and/or nanoparticle containing composition used in combination with a nucleic acid in order to promote uptake of the nucleic acid by a cell.
  • Nucleic acid delivery vehicles include but are not limited to commercially available transfection reagents.
  • nucleobase means a naturally occurring nucleobase or a modified nucleobase.
  • an“unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G).
  • a modified nucleobase is a group of atoms capable of pairing with at least one naturally occurring nucleobase.
  • a universal base is a nucleobase that can pair with any one of the five unmodified nucleobases.
  • “nucleobase sequence” means the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar or intemucleoside linkage modification.
  • nucleoside means a compound comprising a nucleobase and a sugar moiety.
  • the nucleobase and sugar moiety are each, independently, unmodified or modified.
  • modified nucleoside means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety.
  • oligomeric compound means a compound consisting of an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group.
  • oligonucleotide means a strand of linked nucleosides connected via intemucleoside linkages, wherein each nucleoside and intemucleoside linkage may be modified or unmodified. Unless otherwise indicated, oligonucleotides consist of 8-50 linked nucleosides.
  • “modified oligonucleotide” means an oligonucleotide, wherein at least one nucleoside or intemucleoside linkage is modified.
  • unmodified oligonucleotide means an oligonucleotide that does not comprise any nucleoside modifications or intemucleoside modifications.
  • “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. Certain such carriers enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, symps, slurries, suspension and lozenges for the oral ingestion by a subject.
  • a pharmaceutically acceptable carrier or diluent is sterile water; sterile saline; or sterile buffer solution.
  • pharmaceutically acceptable salts means physiologically and pharmaceutically acceptable salts of compounds, such as oligomeric compounds, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • a pharmaceutical composition means a mixture of substances suitable for administering to a subject.
  • a pharmaceutical composition may comprise an antisense compound and a sterile aqueous solution.
  • a pharmaceutical composition shows activity in free uptake assay in certain cell lines.
  • phosphorus moiety means a group of atoms comprising a phosphorus atom.
  • a phosphorus moiety comprises a mono-, di-, or tri-phosphate, or phosphorothioate.
  • prodrug means a therapeutic agent in a form outside the body that is converted to a differentform within the body or cells thereof. Typically conversion of a prodrug within the body is facilitated by the action of an enzymes (e.g., endogenous or viral enzyme) or chemicals present in cells or tissues and/or by physiologic conditions.
  • an enzymes e.g., endogenous or viral enzyme
  • chemicals present in cells or tissues and/or by physiologic conditions.
  • rapalog means a derivative of the molecule, rapamycin.
  • RNAi compound means an antisense compound that acts, at least in part, through RISC or Ago2 to modulate a target nucleic acid and/or protein encoded by a target nucleic acid.
  • RNAi compounds include, but are not limited to double-stranded siRNA, single -stranded RNA (ssRNA), and microRNA, including microRNA mimics.
  • an RNAi compound modulates the amount, activity, and/or splicing of a target nucleic acid.
  • the term RNAi compound excludes antisense oligonucleotides that act through RNase H.
  • the term“single-stranded” in reference to an antisense compound and/or antisense oligonucleotide means an oligomeric compound that is not paired with an additional oligomeric compound to form a duplex.“Self-complementary” in reference to an oligonucleotide means an oligonucleotide that at least partially hybridizes to itself.
  • a compound consisting of one oligomeric compound, wherein the oligonucleotide of the oligomeric compound is self-complementary, is a single-stranded compound.
  • a single- stranded antisense or oligomeric compound may be capable of binding to a complementary oligomeric compound to form a duplex.
  • small molecule means a molecule having a molecular weight equal to or less than 950 Daltons.
  • “sugar moiety” means an unmodified sugar moiety or a modified sugar moiety.
  • “unmodified sugar moiety” means a 2’-OH(H) ribosyl moiety, as found in RNA (an“unmodified RNA sugar moiety”), or a 2’-H(H) moiety, as found in DNA (an“unmodified DNA sugar moiety”).
  • “modified sugar moiety” or“modified sugar” means a modified furanosyl sugar moiety or a sugar surrogate.
  • modified furanosyl sugar moiety means a furanosyl sugar comprising a non- hydrogen substituent in place of at least one hydrogen of an unmodified sugar moiety.
  • a modified furanosyl sugar moiety is a 2’-substituted sugar moiety.
  • modified furanosyl sugar moieties include bicyclic sugars and non-bicyclic sugars.
  • sugar surrogate means a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to another group, such as an intemucleoside linkage, conjugate group, or terminal group in an oligonucleotide.
  • Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to complementary oligomeric compounds or nucleic acids.
  • target nucleic acid means a nucleic acid that an antisense compound affects via hybridization to the target.
  • terminal group means a chemical group or group of atoms that is covalently linked to a terminus of an oligonucleotide.
  • antisense compounds such as certain single -stranded antisense oligonucleotides
  • they must first enter the cell in a way that ultimately facilitates contact with a target nucleic acid. This is particularly important for antisense compounds that are used in the absence of any carrier, nanoparticle, or transfection reagent.
  • Antisense compounds that enter an unproductive uptake pathway do not exhibit antisense activity.
  • Antisense compounds that enter a productive uptake pathway may exhibit antisense activity. At least some productive uptake pathways include one or more steps in which the antisense compound is located inside of an intracellular vesicle.
  • the antisense compound must be released from the vesicle in order to reach its target nucleic acid.
  • Modulating pathways that involve vesicular trafficking may increase antisense compound release or escape from vesicles and/or other steps in productive uptake pathways, ultimately leading to increased antisense activity.
  • Modulating autophagy can increase antisense activity.
  • the autophagy modulation that results in increased antisense activity may occur at one or more steps of the autophagy pathway.
  • some autophagy modulators that increase antisense activity increase the rate of autophagosome formation and/or the number of autophagosomes present in the cell at a given time after autophagy modulation.
  • Autophagosome formation involves multiple steps, including autophagosome nucleation, elongation, sequestration of cytoplasmic contents, and completed formation of an intact autophagosome. See, e.g., Yu et al. Autophagy. Sep 21: 1-9 (2017) Epub ahead of print.
  • Antisense compounds in productive uptake pathways may be contained in some autophagosomes and/or may shuttle between autophagosomes and/or other intracellular vesicles. Accordingly, autophagosomes can fuse temporarily or permanently with endosomes. Eventually, autophagosomes fuse with lysosomes to form autolysosomes. In some cases, autolysosomes are acidified and their contents are destroyed.
  • autolysosomes break apart and revert to separate autophagosomes and lysosomes.
  • Some autophagy modulators that increase antisense activity block the fusion of autophagosomes with lysosomes.
  • antisense compounds that were headed for degradation in an unproductive uptake pathway may be redirected to a productive uptake pathway.
  • autophagy modulators have a specific effect on autophagy. In certain embodiments, autophagy modulators have more than one effect on autophagy. In certain embodiments, autophagy modulators have effects unrelated to autophagy as well as one or more effects on autophagy.
  • the types of effects an autophagy modulator has on a cell may depend on multiple factors, including cell type, basal level of autophagy, and duration of exposure to the autophagy modulator. See, for example, Luu and Luty. Response Profiles of Known Autophagy-Modulators Across Multiple Cell Lines. Enzo Life Sciences Application Note.
  • a method comprising activating autophagy of a cell; and contacting the cell with an antisense compound comprising an antisense oligonucleotide, wherein the nucleobase sequence of the antisense oligonucleotide is complementary to a target nucleic acid.
  • oligonucleotide is at least 80% complementary to the target nucleic acid.
  • oligonucleotide is at least 85% complementary to the target nucleic acid.
  • oligonucleotide is at least 90% complementary to the target nucleic acid.
  • oligonucleotide is at least 95% complementary to the target nucleic acid.
  • oligonucleotide is 100% complementary to the target nucleic acid.
  • oligonucleotide are modified intemucleoside linkages.
  • oligonucleotide are phosphorothioate intemucleoside linkages.
  • oligonucleotide are selected from phosphorothioate and phosphate intemucleoside linkages.
  • each of the modified sugar moieties of the antisense oligonucleotide are independently selected from among modified sugar moieties comprising a 2’-MOE, 2’-0- methyl, cEt, or LNA modification.
  • every nucleoside of the antisense oligonucleotide comprises a modified sugar moiety.
  • each nucleobase of the antisense oligonucleotide is independently selected from among adenine, thymine, guanine, cytosine, and 5-methylcytosine.
  • autophagy modulator is an additional antisense compound comprising an oligonucleotide having a nucleobase sequence that is complementary to a COPB2 transcript.
  • RNAi compound is an siRNA
  • RNAi compound is an siRNA
  • a composition comprising an autophagy modulator and an antisense compound.
  • composition of embodiment 105, wherein the autophagy modulator is rapamycin or a rapalog.
  • composition of embodiment 105, wherein the autophagy modulator is AZD8055.
  • composition of embodiment 105, wherein the autophagy modulator comprises a single stranded, modified oligonucleotide and an autophagy modulator selected from rapamycin, a rapalog, and AZD8055.
  • oligonucleotides oligomeric compounds, and compositions thereof.
  • methods disclosed herein comprise contacting a cell with an oligonucleotide, an oligomeric compound, or a composition thereof.
  • oligonucleotides consist of linked nucleosides.
  • an oligonucleotide is a modified oligonucleotide.
  • the modified oligonucleotide is a modified antisense oligonucleotide.
  • methods disclosed herein comprise contacting a cell with an antisense compound, also referred to as anoligomeric compound, which comprises an oligonucleotide.
  • Oligonucleotides may comprise unmodified nucleosides.
  • oligonucleotides comprise modified nucleosides.
  • modified oligonucleotides comprise at least one modification relative to unmodified RNA or DNA.
  • the modification comprises a modified nucleoside.
  • the modified nucleoside comprises a modified sugar moiety.
  • the modified nucleoside comprises a modified nucleobase.
  • modified nucleosides comprise a modifed sugar moiety and a modified nucleobase.
  • the modification comprises a modified intemucleoside linkage.
  • the modification comprises a modified sugar moiety or a modified intemucleoside linkage.
  • Modified oligoucleotides disclosed herein may comprise a modified sugar moiety, a modified nucleobase, a modified intemucleoside linkage, or any combination thereof.
  • modified sugar moieties are non-bicyclic modified sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain
  • modified sugar moieties are sugar surrogates.
  • Such sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties.
  • modified sugar moieties are non-bicyclic modified furanosyl sugar moieties comprising one or more acyclic substituent, including but not limited to substituents at the 2’, 4’, and/or 5’ positions.
  • the furanosyl sugar moiety is a ribosyl sugar moiety.
  • one or more acyclic substituent of non-bicyclic modified sugar moieties is branched. Examples of 2’-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 2’-F, 2'-OCH 3 (“OMe” or“O-methyl”), and 2'-0(CH 2 ) 2 0CH 3 (“MOE”).
  • 2’ -substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, OCN, CF 3 , OCF 3 , O-Ci-Cio alkoxy, O- C1-C10 substituted alkoxy, O-Ci-Cio alkyl, O-Ci-Cio substituted alkyl, S-alkyl, N(R m )-alkyl, O-alkenyl, S- alkenyl, N(R m )-alkenyl, O-alkynyl, S-alkynyl, N(R m )-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, ( CEE ⁇ SCEE, 0(CH 2 ) 2 0N(R m )(R n ) or 0
  • these 2'-substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl.
  • Examples of 4’-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., WO 2015/106128.
  • Examples of 5’-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 5’-methyl (R or S), 5'- vinyl, and 5’-methoxy.
  • non-bicyclic modified sugars comprise more than one non bridging sugar substituent, for example, 2'-F -5 '-methyl sugar moieties and the modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008/101157 and Rajeev et al., US2013/0203836.).
  • a non-bridging 2’-substituent group selected from: F, OCF3 , OCH3, OCH 2 CH 2 OCH 3 , 0(CH 2 ) 2 SCH 3 , 0(CH 2 ) 2 0N(CH 3 ) 2 , 0(CH 2 ) 2 0(CH 2 ) 2 N(CH 3 ) 2 , and 0CH
  • a 2’-substituted nucleoside or 2’- non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2’-substituent group selected from: F, OCH 3 , and OCH 2 CH 2 OCH 3 .
  • Nucleosides comprising modified sugar moieties may be referred to by the position(s) of the substitution(s) on the sugar moiety of the nucleoside.
  • nucleosides comprising 2’-substituted or 2-modified sugar moieties are referred to as 2’-substituted nucleosides or 2-modified nucleosides.
  • Certain modifed sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicycbc sugar moiety.
  • the bicycbc sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms.
  • the furanose ring is a ribose ring.
  • Examples of such 4’ to 2’ bridging sugar substituents include but are not limited to: 4'-CH 2 -2', 4'- (CH 2 ) 2 -2', 4'-(CH 2 ) 3 -2', 4'-CH 2 -0-2' (“LNA”), 4'-CH 2 -S-2', 4'-(CH 2 ) 2 -0-2' (“ENA”), 4'-CH(CH 3 )-0-2' (referred to as“constrained ethyl” or“cEt” when in the S configuration), 4’-CH 2 -0-CH 2 -2’, 4’-CH 2 -N(R)-2’, 4'-CH(CH 2 0CH 3 )-0-2' (“constrained MOE” or“cMOE”) and analogs thereof (see, e.g., Seth et al., U.S.
  • R a , and R 3 ⁇ 4 is, independently, H, a protecting group, or Ci-Ci 2 alkyl (see, e.g. Imanishi et al., U.S. 7,427,672).
  • such 4’ to 2’ bridges independently comprise from 1 to 4 linked groups independently selected from: -[C(R a )(R b )] n -, -
  • -C(R a ) C(R b )-.
  • x 0, 1, or 2;
  • n 1, 2, 3, or 4;
  • each R a and R 3 ⁇ 4 is, independently, H, a protecting group, hydroxyl, Ci-Ci 2 alkyl, substituted Ci-Ci 2 alkyl, C 2 -Ci 2 alkenyl, substituted C 2 -Ci 2 alkenyl, C 2 -Ci 2 alkynyl, substituted C 2 -Ci 2 alkynyl, C 5 -C 2 o aryl, substituted C 5 -C 2 o aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, NJ
  • bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration.
  • an UNA nucleoside (described herein) may be in the a-U configuration or in the b-D configuration.
  • bicyclic nucleosides include both isomeric configurations.
  • positions of specific bicyclic nucleosides e.g., LNA or cEt
  • they are in the b-D configuration, unless otherwise specified.
  • modified sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5’-substituted and 4’-2’ bridged sugars).
  • modified sugar moieties are sugar surrogates.
  • the oxygen atom of the sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom.
  • such modified sugar moieties also comprise bridging and/or non-bridging substituents as described herein.
  • certain sugar surrogates comprise a 4’-sulfiir atom and a substitution at the 2'- position (see, e.g., Bhat et al., U.S. 7,875,733 and Bhat et al., U.S. 7,939,677) and/or the 5’ position.
  • sugar surrogates comprise rings having other than 5 atoms.
  • a sugar surrogate comprises a six-membered tetrahydropyran (“THP”).
  • THP tetrahydropyran
  • Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified
  • tetrahydropyrans include but are not limited to hexitol nucleic acid (“HNA”), anitol nucleic acid (“ANA”), manitol nucleic acid (“MNA”) (see, e.g., Leumann, CJ. Bioorg. &Med. Chem. 2002, 10, 841-854), fluoro HNA:
  • F-HNA see e.g. Swayze et al., U.S. 8,088,904; Swayze et al., U.S. 8,440,803; Swayze et al., U.S.
  • F-HNA can also be referred to as a F-THP or 3'-fluoro tetrahydropyran), and nucleosides comprising additional modified THP compounds having the formula:
  • Bx is a nucleobase moiety
  • T3 and T4 are each, independently, an intemucleoside linking group linking the modified THP nucleoside to the remainder of an oligonucleotide or one of T3 and T4 is an intemucleoside linking group linking the modified THP nucleoside to the remainder of an oligonucleotide and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3'-terminal group;
  • qi, q2, q3, q4, qs, qe and q 7 are each, independently, H, C 1 -G, alkyl, substituted CrG, alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; and
  • modified THP nucleosides are provided wherein qi, q2, q3, q4, qs, qe and q 7 are each H. In certain embodiments, at least one of qi, q2, q3, q4, qs, qe and q 7 is other than H. In certain embodiments, at least one of qi, q2, q3, q4, qs, qe and q 7 is methyl. In certain embodiments, modified THP nucleosides are provided wherein one of Ri and R2 is F. In certain embodiments, Ri is F and R2 is H, in certain embodiments, Ri is methoxy and R2 is H, and in certain embodiments, Ri is methoxyethoxy and R2 is H.
  • sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom.
  • nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported (see, e.g., Braasch et ah, Biochemistry, 2002, 41, 4503-4510 and Summerton et ah, U.S. 5,698,685; Summerton et ah, U.S. 5,166,315; Summerton et ah, U.S. 5,185,444; and Summerton et ah, U.S. 5,034,506).
  • the term“morpholino” means a sugar surrogate having the following structure:
  • morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure.
  • sugar surrogates are refered to herein as“modifed morpholinos.”
  • sugar surrogates comprise acyclic moieites.
  • nucleosides and oligonucleotides comprising such acyclic sugar surrogates include but are not limited to: peptide nucleic acid (“PNA”), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and nucleosides and oligonucleotides described in Manoharan et al., WO2011/133876.
  • oligonucleotides e.g., antisense oligonucleotides, comprise one or more nucleoside comprising an unmodified nucleobase.
  • modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase.
  • modified nucleobases are selected from: 5-substituted pyrimidines, 6- azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6 substituted purines. In certain embodiments, modified nucleobases are selected from: 2-aminopropyladenine,
  • nucleobases include tricyclic pyrimidines, such as l,3-diazaphenoxazine-2-one, l,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-l,3-diazaphenoxazine-2- one (G-clamp).
  • Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2- pyridone.
  • Further nucleobases include those disclosed in Merigan et al., U.S.
  • nucleosides of modified oligonucleotides may be linked together using any intemucleoside linkage.
  • the two main classes of intemucleoside linking groups are defined by the presence or absence of a phosphorus atom.
  • Modified intemucleoside linkages compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the
  • intemucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Methods of preparation of phosphorous-containing and non- phosphorous-containing intemucleoside linkages are well known to those skilled in the art.
  • Representative intemucleoside linkages having a chiral center include but are not limited to alkylphosphonates and phosphorothioates.
  • Modified oligonucleotides comprising intemucleoside linkages having a chiral center can be prepared as populations of modified oligonucleotides comprising stereorandom intemucleoside linkages, or as populations of modified oligonucleotides comprising phosphorothioate linkages in particular stereochemical configurations.
  • populations of modified oligonucleotides comprise phosphorothioate intemucleoside linkages wherein all of the phosphorothioate intemucleoside linkages are stereorandom.
  • modified oligonucleotides can be generated using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate linkage. Nonetheless, as is well understood by those of skill in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereoconfiguration.
  • populations of modified oligonucleotides are enriched for modified oligonucleotides comprising one or more particular phosphorothioate intemucleoside linkages in a particular, independently selected stereochemical configuration.
  • the particular configuration of the particular phosphorothioate linkage is present in at least 65% of the molecules in the population.
  • the particular configuration of the particular phosphorothioate linkage is present in at least 70% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 80% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 90% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 99% of the molecules in the population.
  • modified oligonucleotides can be generated using synthetic methods known in the art, e.g., methods described in Oka et ak, JACS 125, 8307 (2003), Wan et al. Nuc. Acid. Res. 42, 13456 (2014), and WO 2017/015555.
  • a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one indicated phosphorothioate in the (.S'p) configuration.
  • a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one phosphorothioate in the (Ap) configuration.
  • modified oligonucleotides comprising (/Zp) and/or (.S'p) phosphorothioates comprise one or more of the following formulas, respectively, wherein“B” indicates a nucleobase:
  • chiral intemucleoside linkages of modified oligonucleotides described herein can be stereorandom or in a particular stereochemical configuration.
  • Further neutral intemucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research ; Y.S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral intemucleoside linkages include nonionic linkages comprising mixed N, O, S and CH 2 component parts.
  • modified oligonucleotides comprise one or more modified nucleosides comprising a modified sugar moiety.
  • modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase.
  • modified oligonucleotides comprise one or more modified intemucleoside linkage.
  • the modified, unmodified, and differently modified sugar moieties, nucleobases, and/or intemucleoside linkages of a modified oligonucleotide define a pattern or motif.
  • the patterns of sugar moieties, nucleobases, and intemucleoside linkages are each independent of one another.
  • a modified oligonucleotide may be described by its sugar motif, nucleobase motif and/or intemucleoside linkage motif (as used herein, nucleobase motif describes the modifications to the nucleobases independent of the sequence of nucleobases).
  • oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif.
  • sugar motifs include but are not limited to any of the sugar modifications discussed herein.
  • modified oligonucleotides comprise or consist of a region having a gapmer motif, which is defined by two external regions or“wings” and a central or internal region or“gap.”
  • the three regions of a gapmer motif (the 5’-wing, the gap, and the 3’-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap.
  • the sugar moieties of the nucleosides of each wing that are closest to the gap differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap (i.e., the wing/gap junction).
  • the sugar moieties within the gap are the same as one another.
  • the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap.
  • the sugar motifs of the two wings are the same as one another (symmetric gapmer).
  • the sugar motif of the 5 '-wing differs from the sugar motif of the 3 '-wing (asymmetric gapmer).
  • the wings of a gapmer comprise 1-5 nucleosides. In certain embodiments, each nucleoside of each wing of a gapmer is a modified nucleoside. In certain embodiments, at least one nucleoside of each wing of a gapmer is a modified nucleoside. In certain embodiments, at least two nucleosides of each wing of a gapmer are modified nucleosides. In certain embodiments, at least three nucleosides of each wing of a gapmer are modified nucleosides. In certain embodiments, at least four nucleosides of each wing of a gapmer are modified nucleosides. In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides. In certain embodiments, each nucleoside of the gap of a gapmer is an unmodified 2’-deoxynucleoside. In certain embodiments, at least one nucleoside of the gap of a gapmer is a modified nucleoside.
  • the gapmer is a deoxy gapmer.
  • the nucleosides on the gap side of each wing/gap junction are unmodified 2’-deoxynucleosides and the nucleosides on the wing sides of each wing/gap junction are modified nucleosides.
  • each nucleoside of the gap is an unmodified 2’-deoxynucleoside.
  • each nucleoside of each wing of a gapmer is a modified nucleoside.
  • modified oligonucleotides comprise or consist of a region having a fully modified sugar motif.
  • each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety.
  • each nucleoside of the entire modified oligonucleotide comprises a modified sugar moiety.
  • oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif.
  • a fully modified oligonucleotide is a uniformly modified oligonucleotide.
  • each nucleoside of a uniformly modified comprises the same 2’- modification.
  • the lengths (number of nucleosides) of the three regions of a gapmer may be provided using the notation [# of nucleosides in the 5’-wing] - [# of nucleosides in the gap] - [# of nucleosides in the 3’- wing].
  • a 5-10-5 gapmer consists of 5 linked nucleosides in each wing and 10 linked nucleosides in the gap.
  • that modification is the modification in each sugar moiety of each wing and the gap nucleosides comprise unmodified deoxynucleosides sugars.
  • a 5-10-5 MOE gapmer consists of 5 linked MOE modified nucleosides in the 5’-wing, 10 linked deoxynucleosides in the gap, and 5 linked MOE nucleosides in the 3’-wing.
  • modified oligonucleotides are 5-10-5 MOE gapmers. In certain embodiments, modified oligonucleotides are 5-10-5 MOE gapmers. In certain
  • modified oligonucleotides are 3-10-3 BNA gapmers. In certain embodiments, modified oligonucleotides are 3-10-3 cEt gapmers. In certain embodiments, modified oligonucleotides are 3-10-3 LNA gapmers.
  • oligonucleotides comprising modified and/or unmodified nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or motif.
  • each nucleobase is modified. In certain embodiments, none of the nucleobases are modified.
  • each purine or each pyrimidine is modified.
  • each adenine is modified.
  • each guanine is modified.
  • each thymine is modified.
  • each uracil is modified.
  • each cytosine is modified. In certain embodiments, some or all of the cytosine nucleobases are 5-methylcytosines.
  • modified oligonucleotides such as modified antisense oligonucleotides, comprise a block of modified nucleobases.
  • the block is at the 3’-end of the oligonucleotide. In certain embodiments, the block is within 3 nucleosides of the 3’-end of the
  • the block is at the 5’-end of the oligonucleotide. In certain embodiments, the block is within 3 nucleosides of the 5’-end of the oligonucleotide.
  • one nucleoside comprising a modified nucleobase is in the central gap of an oligonucleotide having a gapmer motif.
  • the sugar moiety of said nucleoside is a 2’-deoxyribosyl moiety.
  • the modified nucleobase is selected from: a 2-thiopyrimidine and a 5-propynepyrimidine.
  • oligonucleotides comprise modified and/or unmodified intemucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or motif.
  • each intemucleoside linkage of a modified oligonucleotide is independently selected from a phosphorothioate intemucleoside linkage and
  • each phosphorothioate intemucleoside linkage is independently selected from a stereorandom phosphorothioate a (.S ' p) phosphorothioate, and a (7Zp) phosphorothioate.
  • the sugar motif of a modified oligonucleotide is a gapmer and the intemucleoside linkages within the gap are all modified.
  • some or all of the intemucleoside linkages in the wings are unmodified phosphodiester intemucleoside linkages.
  • the terminal intemucleoside linkages are modified.
  • the sugar motif of a modified oligonucleotide is a gapmer
  • the intemucleoside linkage motif comprises at least one phosphodiester intemucleoside linkage in at least one wing, wherein the at least one phosphodiester linkage is not a terminal intemucleoside linkage, and the remaining intemucleoside linkages are phosphorothioate intemucleoside linkages.
  • all of the phosphorothioate linkages are stereorandom.
  • all of the phosphorothioate linkages in the wings are (rip) phosphorothioates, and the gap comprises at least one .S ' p .S ' p Rp motif.
  • oligonucleotides are enriched for modified oligonucleotides comprising such intemucleoside linkage motifs.
  • oligonucleotide it is possible to increase or decrease the length of an oligonucleotide without eliminating activity.
  • Woolf et al. Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992
  • a series of oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model.
  • Oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the oligonucleotides were able to direct specific cleavage of the target RNA, albeit to a lesser extent than the oligonucleotides that contained no mismatches.
  • target specific cleavage was achieved using 13 nucleobase oligonucleotides, including those with 1 or 3 mismatches.
  • oligonucleotides can have any of a variety of ranges of lengths.
  • oligonucleotides consist of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number nucleosides in the range.
  • X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X ⁇ Y.
  • oligonucleotides consist of 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15,
  • the above modifications are incorporated into a modified oligonucleotide.
  • such modified oligonucleotides are antisense oligonucleotides.
  • modified oligonucleotides are characterized by their modification motifs and overall lengths. In certain embodiments, such parameters are each independent of one another.
  • each intemucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications.
  • the intemucleoside linkages within the wing regions of a sugar gapmer may be the same or different from one another and may be the same or different from the intemucleoside linkages of the gap region of the sugar motif.
  • sugar gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar
  • an oligonucleotide is described by an overall length or range and by lengths or length ranges of two or more regions (e.g., regions of nucleosides having specified sugar modifications), in such circumstances it may be possible to select numbers for each range that result in an oligonucleotide having an overall length falling outside the specified range. In such circumstances, both elements must be satisfied.
  • a modified oligonucleotide consists if of 15-20 linked nucleosides and has a sugar motif consisting of three regions, A, B, and C, wherein region A consists of 2-6 linked nucleosides having a specified sugar motif, region B consists of 6-10 linked nucleosides having a specified sugar motif, and region C consists of 2-6 linked nucleosides having a specified sugar motif.
  • Such embodiments do not include modified oligonucleotides where A and C each consist of 6 linked nucleosides and B consists of 10 linked nucleosides (even though those numbers of nucleosides are permitted within the requirements for A, B, and C) because the overall length of such oligonucleotide is 22, which exceeds the upper limit of the overall length of the modified oligonucleotide (20).
  • a and C each consist of 6 linked nucleosides and B consists of 10 linked nucleosides (even though those numbers of nucleosides are permitted within the requirements for A, B, and C) because the overall length of such oligonucleotide is 22, which exceeds the upper limit of the overall length of the modified oligonucleotide (20).
  • a description of an oligonucleotide is silent with respect to one or more parameter, such parameter is not limited.
  • a modified oligonucleotide described only as having a gapmer sugar motif without further description may have any
  • Populations of modified oligonucleotides in which all of the modified oligonucleotides of the population have the same molecular formula can be stereorandom populations or chirally enriched populations. All of the chiral centers of all of the modified oligonucleotides are stereorandom in a stereorandom population. In a chirally enriched population, at least one particular chiral center is not stereorandom in the modified oligonucleotides of the population. In certain embodiments, the modified oligonucleotides of a chirally enriched population are enriched for b-D ribosyl sugar moieties, and all of the phosphorothioate intemucleoside linkages are stereorandom.
  • the modified oligonucleotides of a chirally enriched population are enriched for both b-D ribosyl sugar moieties and at least one, particular phosphorothioate intemucleoside linkage in a particular stereochemical configuration.
  • oligonucleotides such as antisense oligonucleotides, are further described by their nucleobase sequence.
  • oligonucleotides have a nucleobase sequence that is complementary to a target oligonucleotide or a target nucleic acid.
  • a region of an oligonucleotide has a nucleobase sequence that is complementary to a target oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid.
  • the nucleobase sequence of a region or entire length of an oligonucleotide is at least 70%, at least 80%, at least 90%, at least 95%, or 100% complementary to the second oligonucleotide or nucleic acid, such as a target nucleic acid.
  • oligomeric compounds which consist of an oligonucleotide (e.g., a modified, unmodified, and/or antisense oligonucleotide) and optionally one or more conjugate groups and/or terminal groups.
  • compositions comprising an oligomeric compound and a pharmaceutically acceptable carrier or diluent.
  • methods comprising contacting a cell with an oligomeric compound or a composition thereof.
  • an oligomeric compound is also an antisense compound.
  • an oligomeric compound is a component of an antisense compound.
  • Conjugate groups consist of one or more conjugate moiety and a conjugate linker which links the conjugate moiety to the oligonucleotide. Conjugate groups may be attached to either or both ends of an oligonucleotide and/or at any internal position. In certain embodiments, conjugate groups are attached to the 2'-position of a nucleoside of a modified oligonucleotide. In certain embodiments, conjugate groups that are attached to either or both ends of an oligonucleotide are terminal groups. In certain such embodiments, conjugate groups or terminal groups are attached at the 3’ and/or 5’-end of oligonucleotides. In certain such embodiments, conjugate groups (or terminal groups) are attached at the 3’-end of
  • conjugate groups are attached near the 3’-end of oligonucleotides. In certain embodiments, conjugate groups (or terminal groups) are attached at the 5’-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 5’-end of oligonucleotides.
  • terminal groups include but are not limited to conjugate groups, capping groups, phosphate moieties, protecting groups, abasic nucleosides, modified or unmodified nucleosides, and two or more nucleosides that are independently modified or unmodified.
  • oligonucleotides are covalently attached to one or more conjugate groups.
  • conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance.
  • conjugate groups impart a new property on the attached oligonucleotide, e.g., fluorophores or reporter groups that enable detection of the oligonucleotide.
  • conjugate groups and conjugate moieties have been described previously, for example: cholesterol moiety (Letsinger et ah, Proc. Natl. Acad. Sci.
  • a phospholipid e.g., di-hexadecyl-rac -glycerol or triethyl-ammonium l,2-di-0-hexadecyl-rac-glycero-3- H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,
  • Conjugate moieties include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates (e.g., GalNAc), vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.
  • intercalators include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates (e.g., GalNAc), vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, bio
  • a conjugate moiety comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (.S')-(+)-pranoprofcn carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a
  • an active drug substance for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (.S')-(+)-pranoprofcn carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a
  • benzothiadiazide chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • Conjugate moieties are attached to oligonucleotides through conjugate linkers.
  • the conjugate linker is a single chemical bond (i.e., the conjugate moiety is attached directly to an oligonucleotide through a single bond).
  • a conjugate moiety is attached to an oligonucleotide via a more complex conjugate linker comprising one or more conjugate linker moieities, which are sub-units making up a conjugate linker.
  • the conjugate linker comprises a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units such as ethylene glycol, nucleosides, or amino acid units.
  • a conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises groups selected from alkyl, amino, oxo, amide and ether groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker includes at least one neutral linking group.
  • conjugate linkers are bifunctional linking moieties, e.g., those known in the art to be useful for attaching conjugate groups to parent compounds, such as the oligonucleotides provided herein.
  • a bifunctional linking moiety comprises at least two functional groups. One of the functional groups is selected to bind to a particular site on a parent compound and the other is selected to bind to a conjugate group. Examples of functional groups used in a bifunctional linking moiety include but are not limited to electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups.
  • bifunctional linking moieties comprise one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.
  • conjugate linkers include but are not limited to pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane- l-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA).
  • ADO 8-amino-3,6-dioxaoctanoic acid
  • SMCC succinimidyl 4-(N-maleimidomethyl) cyclohexane- l-carboxylate
  • AHEX or AHA 6-aminohexanoic acid
  • conjugate linkers include but are not limited to substituted or unsubstituted Ci- Cio alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
  • conjugate linkers comprise 1-10 linker-nucleosidesln certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments such linker-nucleosides comprise a modified sugar moiety. In certain embodiments, linker-nucleosides are unmodified. In certain embodiments,
  • linker-nucleosides comprise an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine.
  • a cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N -benzoyl-5 - methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is typically desirable for linker-nucleosides to be cleaved from the oligomeric compound after it reaches a target tissue.
  • linker-nucleosides are typically linked to one another and to the remainder of the oligomeric compound through cleavable bonds.
  • cleavable bonds are phosphodiester bonds.
  • linker-nucleosides are not considered to be part of the oligonucleotide. Accordingly, in embodiments in which an oligomeric compound comprises an oligonucleotide consisting of a specified number or range of linked nucleosides and/or a specified percent complementarity to a reference nucleic acid and the oligomeric compound also comprises a conjugate group comprising a conjugate linker comprising linker-nucleosides, those linker-nucleosides are not counted toward the length of the oligonucleotide and are not used in determining the percent complementarity of the oligonucleotide for the reference nucleic acid.
  • an oligomeric compound may comprise (1) a modified oligonucleotide consisting of 8-30 nucleosides and (2) a conjugate group comprising 1-10 linker-nucleosides that are contiguous with the nucleosides of the modified oligonucleotide.
  • the total number of contiguous linked nucleosides in such an oligomeric compound is more than 30.
  • an oligomeric compound may comprise a modified oligonucleotide consisting of 8-30 nucleosides and no conjugate group. The total number of contiguous linked nucleosides in such an oligomeric compound is no more than 30.
  • conjugate linkers comprise no more than 10 linker-nucleosides.
  • conjugate linkers comprise no more than 5 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 3 linker- nucleosides. In certain embodiments, conjugate linkers comprise no more than 2 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 1 linker-nucleoside.
  • a conjugate group it is desirable for a conjugate group to be cleaved from the oligonucleotide.
  • oligomeric compounds comprising a particular conjugate moiety are better taken up by a particular cell type, but once the oligomeric compound has been taken up, it is desirable that the conjugate group be cleaved to release the unconjugated or parent oligonucleotide.
  • certain conjugate linkers may comprise one or more cleavable moieties.
  • a cleavable moiety is a cleavable bond.
  • a cleavable moiety is a group of atoms comprising at least one cleavable bond.
  • a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds.
  • a cleavable moiety is selectively cleaved inside a cell or subcellular compartment, such as a lysosome.
  • a cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases.
  • a cleavable bond is selected from among: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In certain embodiments, a cleavable bond is one or both of the esters of a phosphodiester. In certain embodiments, a cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a phosphate linkage between an oligonucleotide and a conjugate moiety or conjugate group.
  • a cleavable moiety comprises or consists of one or more linker-nucleosides.
  • the one or more linker-nucleosides are linked to one another and/or to the remainder of the oligomeric compound through cleavable bonds.
  • such cleavable bonds are unmodified phosphodiester bonds.
  • a cleavable moiety is 2'-deoxy nucleoside that is attached to either the 3' or 5 '-terminal nucleoside of an oligonucleotide by a phosphate intemucleoside linkage and covalently attached to the remainder of the conjugate linker or conjugate moiety by a phosphate or phosphorothioate linkage.
  • the cleavable moiety is 2'- deoxyadenosine.
  • compounds of the invention are single -stranded.
  • oligomeric compounds are paired with an additionaloligonucleotide or oligomeric compound to form a duplex, which is double-stranded.
  • oligomeric compounds comprise one or more terminal groups.
  • oligomeric compounds comprise a stabilized 5’-phophate.
  • Stabilized 5’-phosphates include, but are not limited to 5’-phosphanates, including, but not limited to 5’-vinylphosphonates.
  • terminal groups comprise one or more abasic nucleosides and/or inverted nucleosides.
  • terminal groups comprise one or more 2’-linked nucleosides. In certain such embodiments, the 2’-linked nucleoside is an abasic nucleoside.
  • oligomeric compounds described herein comprise an oligonucleotide, having a nucleobase sequence complementary to that of a target nucleic acid.
  • an oligomeric compound is paired with a second oligomeric compound to form an oligomeric duplex.
  • Such oligomeric duplexes comprise a first oligomeric compound having a region complementary to a target nucleic acid and a second oligomeric compound having a region complementary to the first oligomeric compound.
  • the first oligomeric compound of an oligomeric duplex comprises or consists of (1) a modified or unmodified oligonucleotide and optionally a conjugate group and (2) a second modified or unmodified oligonucleotide and optionally a conjugate group.
  • Either or both oligomeric compounds of an oligomeric duplex may comprise a conjugate group.
  • the oligonucleotides of each oligomeric compound of an oligomeric duplex may include non-complementary overhanging nucleosides.
  • oligomeric compounds and oligomeric duplexes are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity; such oligomeric compounds and oligomeric duplexes are antisense compounds.
  • antisense compounds have antisense activity when they reduce or inhibit the amount or activity of a target nucleic acid by 25% or more in the standard cell assay. In certain embodiments, antisense compounds selectively affect one or more target nucleic acid.
  • Such antisense compounds comprise a nucleobase sequence that hybridizes to one or more target nucleic acid, resulting in one or more desired antisense activity and does not hybridize to one or more non-target nucleic acid or does not hybridize to one or more non-target nucleic acid in such a way that results in significant undesired antisense activity.
  • hybridization of an antisense compound to a target nucleic acid results in recruitment of a protein that cleaves the target nucleic acid.
  • certain antisense compounds result in RNase H mediated cleavage of the target nucleic acid.
  • RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex.
  • RNA:DNA duplex need not be unmodified DNA.
  • described herein are antisense compounds that are sufficiently “DNA-like” to elicit RNase H activity.
  • one or more non-DNA-like nucleoside in the gap of a gapmer is tolerated.
  • an antisense compound or a portion of an antisense compound is loaded into an RNA-induced silencing complex (RISC), ultimately resulting in cleavage of the target nucleic acid.
  • RISC RNA-induced silencing complex
  • certain antisense compounds result in cleavage of the target nucleic acid by Argonaute.
  • Antisense compounds that are loaded into RISC are RNAi compounds. RNAi compounds may be double- stranded (siRNA) or single -stranded (ssRNA).
  • hybridization of an antisense compound to a target nucleic acid does not result in recruitment of a protein that cleaves that target nucleic acid. In certain embodiments, hybridization of the antisense compound to the target nucleic acid results in alteration of splicing of the target nucleic acid. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in inhibition of a binding interaction between the target nucleic acid and a protein or other nucleic acid. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in alteration of translation of the target nucleic acid.
  • Antisense activities may be observed directly or indirectly.
  • observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid, a change in the ratio of splice variants of a nucleic acid or protein and/or a phenotypic change in a cell or subject.
  • antisense compounds comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid.
  • the target nucleic acid is an endogenous RNA molecule.
  • the target nucleic acid encodes a protein.
  • the target nucleic acid is a mRNA.
  • the target region is entirely within an exon.
  • the target region spans an exon/exon junction.
  • antisense compounds are at least partially complementary to more than one target nucleic acid.
  • Gautschi et al J. Natl. Cancer Inst. 93:463-471, March 2001
  • this oligonucleotide demonstrated potent anti tumor activity in vivo. Maher and Dolnick (Nuc. Acid. Res.
  • oligonucleotides are complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99%, 95%, 90%, 85%, or 80% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide and comprise a region that is 100% or fully complementary to a target nucleic acid. In certain embodiments, the region of full complementarity is from 6 to 20, 10 to 18, or 18 to 20 nucleobases in length.
  • oligonucleotides comprise one or more mismatched nucleobases relative to the target nucleic acid.
  • antisense activity against the target is reduced by such mismatch, but activity against a non-target is reduced by a greater amount.
  • selectivity of the oligonucleotide is improved.
  • the mismatch is specifically positioned within an oligonucleotide having a gapmer motif.
  • the mismatch is at position 1, 2, 3, 4, 5, 6, 7, or 8 from the 5’-end of the gap region.
  • the mismatch is at position 9, 8, 7, 6, 5, 4, 3, 2, 1 from the 3’-end of the gap region.
  • the mismatch is at position 1, 2, 3, or 4 from the 5’-end of the wing region.
  • the mismatch is at position 4, 3, 2, or 1 from the 3’-end of the wing region.
  • compositions comprising one or more oligomeric compounds, and optionally an autophagy modulator.
  • the one or more oligomeric compounds each consists of a modified oligonucleotide.
  • the one or more oligomeric compounds each consists of a modified oligonucleotide.
  • a pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier.
  • a pharmaceutical composition comprises or consists of a sterile saline solution and one or more oligomeric compound.
  • the sterile saline is pharmaceutical grade saline.
  • a pharmaceutical composition comprises or consists of one or more oligomeric compound and sterile water.
  • the sterile water is pharmaceutical grade water.
  • a pharmaceutical composition comprises or consists of one or more oligomeric compound and phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • the sterile PBS is pharmaceutical grade PBS.
  • a pharmaceutical composition comprises or consists of one or more oligomeric compound and artificial cerebrospinal fluid.
  • the artificial cerebrospinal fluid is pharmaceutical grade.
  • a pharmaceutical composition comprises a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, a pharmaceutical composition consists of a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, a pharmaceutical composition consists essentially of a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, the artificial cerebrospinal fluid is pharmaceutical grade.
  • compositions comprise one or more oligomeric compound and one or more excipients.
  • excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.
  • oligomeric compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations.
  • compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
  • compositions comprising an oligomeric compound encompass any pharmaceutically acceptable salts of the oligomeric compound, esters of the oligomeric compound, or salts of such esters.
  • pharmaceutical compositions comprising oligomeric compounds comprising one or more oligonucleotide upon administration to a subject, including a human, are capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • the disclosure is also drawn to pharmaceutically acceptable salts of oligomeric compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
  • Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
  • prodrugs comprise one or more conjugate group attached to an oligonucleotide, wherein the conjugate group is cleaved by endogenous nucleases within the body.
  • Lipid moieties have been used in nucleic acid therapies in a variety of methods.
  • the nucleic acid such as an oligomeric compound, is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids.
  • DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
  • compositions comprise a delivery system.
  • delivery systems include, but are not limited to, liposomes and emulsions.
  • Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds.
  • certain organic solvents such as dimethylsulfoxide are used.
  • compositions comprise one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types.
  • pharmaceutical compositions include liposomes coated with a tissue-specific antibody.
  • compositions comprise a co-solvent system.
  • co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • co-solvent systems are used for hydrophobic compounds.
  • VPD co-solvent system is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant
  • Polysorbate 80TM and 65% w/v polyethylene glycol 300 The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80TM; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
  • compositions are prepared for oral administration.
  • pharmaceutical compositions are prepared for buccal administration.
  • a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, intrathecal (IT), intracerebroventricular (ICV), etc.).
  • a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives).
  • injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like.
  • compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi -dose containers.
  • Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.
  • methods provided herein comprise inducing autophagy in a cell and administering or contacting the cell with an antisense compound. In certain embodiments, methods provided herein comprise enhancing or increasing autophagy in a cell and administering or contacting the cell with an antisense compound.
  • methods disclosed herein comprise contacting a cell with an antisense oligonucleotide capable of hybridizing to a target nucleic acid in the cell and activating autophagy in the cell, thereby modifying an amount of the target nucleic acid in the cell.
  • methods disclosed herein comprise reducing an amount of a target nucleic acid in a cell.
  • activating autophagy and contacting the cell with the antisense oligonucleotide reduces the amount of the target nucleic acid in the cell to a greater extent than in the absence of activating autophagy.
  • methods comprise reducing expression of the target nucleic acid.
  • activating autophagy and contacting the cell with the antisense oligonucleotide reduces the expression of the target nucleic acid in the cell to a greater extent than in the absence of activating autophagy.
  • methods disclosed herein comprise increasing an amount of a target nucleic acid in a cell.
  • activating autophagy and contacting the cell with the antisense oligonucleotide increases the amount of the target nucleic acid in the cell to a greater extent than in the absence of activating autophagy.
  • methods comprise increasing expression of the target nucleic acid.
  • activating autophagy and contacting the cell with the antisense oligonucleotide increases the expression of the target nucleic acid in the cell to a greater extent than in the absence of activating autophagy.
  • methods comprise modifying splicing of a target nucleic acid.
  • activating autophagy and contacting the cell with the antisense oligonucleotide increases modifies splicing of the target nucleic acid in the cell to a greater extent than in the absence of activating autophagy.
  • methods comprise fasting a subject and administering an antisense compound, or a composition thereof, to the subject.
  • fasting refers to caloric deprivation.
  • a subject that is fasting may still consume water and non-caloric fluids.
  • inducing, enhancing or increasing autophagy comprises starving a cell in vitro.
  • methods comprise fasting a subject before administering the antisense compound to the subject.
  • methods comprise fasting the subject for at least 16 hours before administering the antisense compound.
  • methods comprise fasting a subject after administering an antisense compound to the subject.
  • inducing, enhancing or increasing autophagy comprises starving a cell in vitro.
  • starving the cell comprises culturing the cell in a starvation media.
  • inducing, enhancing or increasing autophagy in a cell comprises restricting and/or increasing nutrients, thereby stimulating autophagy in the cell.
  • inducing, enhancing or increasing autophagy comprises exposing the cell to a ratio of nutrients that stimulates autophagy in the cell.
  • the cell may be present in a subject and the subject may be fed a ketogenic diet, thereby inducing autophagy in cells of the subject.
  • a ketogenic diet may be defined by percentages of caloric intake obtained from fat, protein and carbohydrates.
  • At least 90% of caloric intake is from fat. In certain embodiments, at least 85% of caloric intake is from fat. In certain embodiments, at least 80% of caloric intake is from fat. In certain embodiments, at least 75% of caloric intake is from fat.
  • methods comprise exposing a subject to a ketogenic diet and administering an antisense compound to the subject. In certain embodiments, methods comprise exposing a subject to a ketogenic diet before administering an antisense compound to the subject. In certain embodiments, methods comprise exposing a subject to a ketogenic diet after administering an antisense compound to the subject. In certain embodiments, methods comprise administering an antisense compound to a subject while the subject is on a ketogenic diet. In certain embodiments, methods comprise administering the antisense compound to the subject, wherein the subject has followed a ketogenic diet for at least one day, at least two days, at least three days, or at least five days before the administering.
  • methods comprise administering the antisense compound to the subject, wherein the subject has followed a ketogenic diet for at least one week before the administering. In certain embodiments, methods comprise administering the antisense compound to the subject, wherein the subject has followed a ketogenic diet for at least two weeks, at least three weeks, or at least five weeks before the administering. In certain embodiments, the subject follows a ketogenic diet for at least one day, at least two days, at least three days, or at least five days after the administering. In certain embodiments, the subject follows a ketogenic diet for at least one week after the administering. In certain embodiments, the subject follows a ketogenic diet for at least two weeks, at least three weeks, or at least four weeks after the administering.
  • methods provided herein comprise contacting a cell with an antisense compound and an autophagy modulator, wherein the autophagy modulator increases the activity of the antisense compound in the cell relative to the activity of the antisense compound in the cell in the absence of the autophagy modulator.
  • the cell is in vivo and methods comprise administering the autophagy modulator and antisense compound to the subject.
  • methods comprise adeministering an antisense compound and one or more autophagy modulators in combination.
  • methods comprise administering an antisense compound and an autophagy modulator simultaneously.
  • methods comprise administering an antisense compound and an autophagy modulator separately.
  • methods comprise administering an antisense compound and an autophagy modulator sequentially.
  • the antisense compound and the autophagy modulator are formulated as a fixed dose combination product. In other embodiments, they are provided to the patient as separate units which can then either be taken simultaneously or serially
  • administration of the autophagy modulator and antisense compound permits use of a lower dosage of the antisense compound than would be required to achieve a therapeutic or prophylactic effect if the antisense compound were administered alone.
  • co administration of the antisense compound and the autophagy modulator permits use of a lower dosage of the antisense compound to achieve a therapeutic result, wherein the lower dosage is lower than a comparative dosage of the antisense compound necessary to achieve the therapeutic result when the antisense compound is used without the autophagy modulator.
  • methods comprise inducing or enhancing autophagy via fasting or nutrient restriction.
  • inducing or enhancing autophagy permits use of a lower dose of the antisense compound to achieve a therapeutic result relative to a comparative dose of the antisense compound when the compound is administeredin the absence of inducing or enhancing autophagy.
  • methods comprise co-administering an antisense compound comprising or consisting of an antisense oligonucleotide with one or more autophagy modulators.
  • the antisense compound and one or more autophagy modulators are administered
  • an antisense compound comprising or consisting of an antisense oligonucleotide and one or more autophagy modulators are administered to a subject sequentially. In certain such embodiments, the antisense compound and one or more autophagy modulators are administered at different times. In certain embodiments, the antisense compound is administered before the one or more autophagy modulators. In certain embodiments, the antisense compound is administered after the one or more autophagy modulators.
  • methods comprise administering the antisense compound and one or more autophagy modulators together in a single formulation.
  • compositions disclosed herein comprise an autophagy modulator and an antisense oligonucleotide in a single formulation.
  • methods comprise administering the antisense compound and one or more autophagy modulators, wherein the antisense compound and one or more autophagy modulators are prepared as separate formulations.
  • compositions and methods disclosed herein comprise an autophagy modulator, or a use thereof.
  • the autophagy modulator is a small molecule.
  • the autophagy modulator is an autophagy activator.
  • the autophagy activator induces autophagosome formation. In certain embodiments, the autophagy activator induces autophagosome nucleation. In certain embodiments, the autophagy activator induces autophagosome elongation. In certain embodiments, the autophagy activator increases the number of autophagosomes in the cell. In certain embodiments, the autophagy activator increases expression of an autophagy pathway component. In certain embodiments, the autophagy activator increases autophagosome formation relative to that expected at a basal level of autophagy. In certain embodiments, the autophagy activator increases autophagosome nucleation relative to a basal level of autophagy in the absence of the autophagy activator.
  • the autophagy activator increases autophagosome elongation relative to a basal level of autophagy in the absence of the autophagy activator. In certain embodiments, the autophagy activator increases the number of autophagic vesicles in the cell relative to a basal level of autophagy in the absence of the autophagy activator. In certain embodiments, the autophagy activator increases the number of autophagosomes in the cell relative to a basal level of autophagy in the absence of the autophagy activator.
  • compositions and methods disclosed herein comprise an autophagy modulator, or a use thereof, wherein the autophagy activator is an mTor inhibitor.
  • the mTor inhibitor is capable of inhibiting mTor activity in the cell.
  • mTor activity comprises kinase activity, protein binding activity, or a comabination thereof.
  • the mTor inhibitor comprises rapamycin.
  • the mTor inhibitor is rapamycin.
  • the mTor inhibitor comprises a rapalog.
  • the rapalog is temsirolimus.
  • the rapalog is everolimus. In certain embodiments, the rapalog is ridaforolimus.
  • the autophagy activator is an ATP -competitive mTor inhibitor.
  • Non-limiting examples of ATP -competitive mTor inhibitors are OSI-027, AZD8055, AZD2014, and INK128.
  • the autophagy activator is AZD8055. In certain embodiments, the autophagy activator selectively inhibits mTor over other kinases. In certain embodiments, the autophagy activator selectively inhibits mTor over PI3 kinase. In certain embodiments, the autophagy activator inhibits mTor and at least one other kinase. In certain embodiments, the autophagy activator is not a PI3 kinase inhibitor. In certain embodiments, the autophagy activator is PI- 103. In certain embodiments, the autophagy activator is PP242.
  • the autophagy activator increases expression of LC3. In certain embodiments, the autophagy activator increases expression of LC3-II. In certain embodiments, the autophagy activator increases the amount of LC3-II. In certain embodiments, the autophagy activator increases expression of Rab7, LAMP-2, Atg7, ULK1, ULK2, Atg5, Beclin, or c-Jun. In certain embodiments, compositions and methods disclosed herein comprise an autophagy modulator, or a use thereof, wherein the autophagy activator blocks fusion of autophagosomes to lysosomes.
  • the autophagy modulator increases the number of autophagic vesicles in the cell relative to that expected at a basal level of autophagy. In certain embodiments, the autophagy activator increases the number of autophagosomes in the cell relative to that expected at a basal level of autophagy. In certain embodiments, the autophagy modulator is Vinblastine. In certain embodiments, the autophagy modulator is Bafilomycin Al. In certain embodiments, the autophagy modulator is a COPB2 inhibitor. In certain embodiments, the autophagy modulator is capable of inhibiting COPB2.
  • RNA nucleoside comprising a 2’-OH sugar moiety and a thymine base
  • RNA thymine (methylated uracil) in place of a uracil of RNA
  • nucleic acid sequences provided herein are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases.
  • an oligomeric compound having the nucleobase sequence“ATCGATCG” encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence“AUCGAUCG” and those having some DNA bases and some RNA bases such as“AUCGATCG” and oligomeric compounds having other modified nucleobases, such as“AT m CGAUCG,” wherein '"C indicates a cytosine base comprising a methyl group at the 5-position.
  • Certain compounds described herein e.g., antisense oligonucleotides
  • Compounds provided herein that are drawn or described as having certain stereoisomeric configurations include only the indicated compounds.
  • Compounds provided herein that are drawn or described with undefined stereochemistry include all such possible isomers, including their racemic and optically pure forms. All tautomeric forms of the compounds provided herein are included unless otherwise indicated.
  • the compounds described herein include variations in which one or more atoms are replaced with a non-radioactive isotope or radioactive isotope of the indicated element.
  • compounds herein that comprise hydrogen atoms encompass all possible deuterium substitutions for each of the 3 ⁇ 4 hydrogen atoms.
  • Isotopic substitutions encompassed by the compounds herein include but are not limited to: 2 H or 3 H in place of 3 ⁇ 4, 13 C or 14 C in place of 12 C, 15 N in place of 14 N, 17 0 or 18 0 in place of 16 0, and 33 S, 34 S, 35 S, or 36 S in place of 32 S.
  • non-radioactive isotopic substitutions may impart new properties on the oligomeric compound that are beneficial for use as a therapeutic or research tool.
  • radioactive isotopic substitutions may make the compound suitable for research or diagnostic purposes such as imaging.
  • the following examples demonstrate autophagy enhancement of antisense activity in a wide variety of cell types (mouse and human) with a variety of antisense compounds and a variety of autophagy modulating compounds to demonstrate that the effects of autophagy modulation on antisense acitivity are not limited to any one of these parameters.
  • a wide variety of autophagy activating compounds enhanced antisense oligonucleotide activity in primary cells, embryonic fibroblasts and cell lines from rodents and humans.
  • Cell types in which autophagy modulators enhanced antisense oligonucleotide activity included cells originating from liver, skin, neuronal tissue, cervical epithelium, connective tissue, and lung, as well as mouse embryonic fibroblasts, and human fibroblasts.
  • the following examples demonstrate that the antisense activity enhancing effects of autophagy activating compounds are observed in vivo, and that conditions that activate autophagy, such as fasting and ketogenic diet enhance antisense activitiy in mice. Further, the following examples demonstrate that antisense oligonucleotide can be chemically modified in various ways without abolishing the antisense activity enhancing effects of autophagy activation. Several antisense oligonucleotides were 5-10-5 MOE gapmers, while others were 3-10-3 cEt gapmers, and still others comprised a GalNAc moiety.
  • Example 1 Effect of modulation of autophagy on antisense activity in vitro , single dose
  • MHT cells (a mouse hepatocellular carcinoma cell line) were plated at 20,000 cells/well and treated with DMSO (control) or an autophagy modulator for 24 hours at the concentrations indicated in Table 2 below.
  • 3-MA, BafAl, AZD8055, and leupeptin are autophagy activators.
  • Chloroquine is an autophagy inhibitor.
  • 10 mM of a modified control oligonucleotide or of a modified antisense oligonucleotide was added by free uptake for ten hours.
  • the modified oligonucleotides shown in Table 1 below, are 3-10-3 cEt gapmers in which every intemucleoside linkage is a phosphorothioate intemucleoside linkage.
  • the three 5’-terminal nucleosides and the three 3’- terminal nucleosides of each oligonucleotide each comprise a cEt modified sugar moiety.
  • the remaining ten nucleosides of each oligonucleotide each comprise an unmodified, 2’-deoxy sugar moiety. All of the cytosines had a methyl at the 5 position.
  • RNA levels of Malat-l shown in Table 4 and markers of autophagy, (Rab7, LAMP2, Atg7, ULK1, ULK2, Atg5, Beclin, and c-Jun), shown in Table 5. Results were normalized to cyclophilin A levels.
  • Malat-l RNA levels after treatment with an antisense oligonucleotide and an autophagy modulator were normalized to Malat-l RNA levels in cells treated with control Compound No. 549148 and the same autophagy modulator.
  • Example 2 Effect of modulation of autophagy on antisense activity in vitro, multiple doses of antisense oligonucleotides
  • antisense oligonucleotides in the following example have a different pattern of chemical modifications (5-10-5 MOE gapmers) than those used in Example 1 (3-10-3 cEt gapmers).
  • MHT cells were plated at 20,000 cells/well and treated with DMSO (control) or an autophagy modulator for 24 hours, at the concentrations indicated Table 7 below.
  • control or antisense oligonucleotides were added at 1 mM, 2.5 mM, 5 mM, or 10 mM by free uptake for ten hours.
  • the modified oligonucleotides, shown in Table 6 below, are 5-10-5 MOE gapmers in which every intemucleoside linkage is a phosphorothioate intemucleoside linkage.
  • the five 5’-terminal nucleosides and the five 3’-terminal nucleosides of each oligonucleotide each comprise a 2’-MOE modified sugar moiety.
  • the remaining ten nucleosides of each oligonucleotide each comprise an unmodified, 2’-deoxy sugar moiety. All of the cytosines had a methyl at the 5 position.
  • RT-qPCR analysis was performed to detect RNA levels of Malat-l using primer probe set 15877 (described in Example 1).
  • primer probe set 15877 described in Example 1.
  • Malat-l RNA levels after treatment with an antisense oligonucleotide and an autophagy modulator were normalized to Malat-l RNA levels in cells treated with control Compound No. 141923 and the same autophagy modulator.
  • Each table below represents results from an individual assay.
  • Tables 8-12 show the results of five experiments performed separately. All results demonstrate that induction of autophagy by 3-MA, BafAl, AZD8055, rapamycin, VPS34-IN2, vinblastine, and leupeptin significantly enhanced modified oligonucleotide activity of MOE gapmers in MHT cells relative to cells treated with control DMSO. In contrast, inhibitors of autophagy (wortmannin, E64D, and chloroquine) reduced activity of modified oligonucleotide relative to cells treated with control DMSO. These results were observed regardless of antisense oligonucleotide dose and specific autophagy modulator. Together the results of Example 1 and Example 2 suggest that autophagy modulators enhance the activity of antisense oligonucleotides regardless of antisense oligonucleotide chemical modifications.
  • Example 3 Effect of modulation of autophagy on antisense activity in vitro in MHT cells (mouse hepatocellular carcinoma cell model), multiple dose
  • MHT cells were plated at 20,000 cells/well and treated with DMSO (control) or an autophagy modulator for 24 hours, at the concentrations indicated in Example 1.
  • DMSO control
  • an autophagy modulator for 24 hours, at the concentrations indicated in Example 1.
  • a modified control oligonucleotide or modified antisense oligonucleotide was added at 2.5 mM, 5 mM, or 10 mM by free uptake for ten hours, as indicated in the tables below.
  • the modified oligonucleotides, shown in Table 13 below, are 5-10-5 MOE gapmers in which every intemucleoside linkage is a phosphorothioate intemucleoside linkage.
  • each oligonucleotide comprises a 2’-MOE modified sugar moiety.
  • the remaining ten nucleosides of each oligonucleotide each comprise an unmodified, 2’-deoxy sugar moiety. All the cytosines had a methyl at the 5 position.
  • Table 13 Modified oligonucleotides
  • RT-qPCR analysis was performed to detect mRNA levels of SRB1 using primer probe set 15299 (forward sequence: TGACAACGACACCGTGTCCT, SEQ ID NO:27; reverse sequence:
  • results were normalized to cyclophilin A levels.
  • SRB 1 mRNA levels after treatment with an antisense oligonucleotide and an autophagy modulator were normalized to SRB1 mRNA levels in cells treated with control Compound No. 141923 and the same autophagy modulator.
  • Results presented in Table 14 demonstrate that induction of autophagy significantly enhanced activity of modified oligonucleotides (MOE gapmers) targeting SRB1 in MHT cells. In contrast, inhibition of autophagy reduced antisense activity.
  • MOE gapmers modified oligonucleotides
  • Example 4 Effect of modulation of autophagy on antisense activity in HeLa cells (human cervical cancer cell line), multiple dose
  • Examples 1-3 demonstrate that activating autophagy in a liver cell line enhances antisense activity against multiple targets regardless of antisense oligonucleotide chemical modifications. To query whether these results were cell-type specific or species specific, similar experiments were repeated in a human epithelial cell line derived from a cervical cancer cells (HeLa).
  • HeLa cervical cancer cells
  • HeLa cells were plated at 10,000 cells/well and treated with DMSO (control) or an autophagy modulator for 24 hours, at the concentrations indicated in Example 1 or in the tables below.
  • control or antisense oligonucleotides were added at 2.5 mM, 5 pM, or 10 pM by free uptake for ten hours, as indicated in the tables below.
  • the modified oligonucleotides, shown in Table 15 below, are 5-10-5 MOE gapmers in which every intemucleoside linkage is a
  • each oligonucleotide comprises a 2’-MOE modified sugar moiety.
  • the remaining ten nucleosides of each oligonucleotide each comprise an unmodified, 2’-deoxy sugar moiety. All of the cytosines had a methyl at the 5 position.
  • RT-qPCR analysis was performed to detect RNA levels of Malat-l using primer probe set RTS2736 (forward sequence AAAGCAAGGTCTCCCCACAAG, designated herein as SEQ ID NO: 32; reverse sequence TGAAGGGTCTGTGCTAGATCAAAA, designated herein as SEQ ID NO: 33; probe sequence TGCCACATCGCCACCCCGT, designated herein as SEQ ID NO: 34).
  • Results were normalized to cyclophilin A levels, measured using human cyclophilin A primer probe set HTS3936 (forward sequence GCCATGGAGCGCTTTGG, designated herein as SEQ ID NO: 35; reverse sequence
  • TCCACAGTCAGCAATGGTGATC designated herein as SEQ ID NO: 36; probe sequence
  • TCCAGGAATGGCAAGACCAGCAAGA TCCAGGAATGGCAAGACCAGCAAGA, designated herein as SEQ ID NO: 37.
  • Malat-l RNA levels after treatment with an antisense oligonucleotide and an autophagy modulator were normalized to Malat-l RNA levels in cells treated with control Compound No. 141923.
  • Example 5 Effect of modulation of autophagy on antisense activity in vitro in epidermoid carcinoma
  • A431 cells were plated at 16,300 cells/well and treated with DMSO (control) or an autophagy modulator for 24 hours, at the concentrations indicated in Example 1.
  • DMSO control
  • an autophagy modulator for 24 hours, at the concentrations indicated in Example 1.
  • a modified control oligonucleotide Compound No. 141923 described in examples above
  • modified antisense oligonucleotide Compound No. 395254 described in examples above
  • RT-qPCR analysis was performed to detect RNA levels of Malat-l using primer probe set RTS2736.
  • Results were normalized to cyclophilin A levels (measured using HTS3936).
  • Results were normalized to Malat-l RNA levels in cells treated with control Compound No. 141923.
  • Each table below represents results from an individual assay.
  • Example 6 Effect of modulation of autophagy prior to antisense oligonucleotide treatment
  • MHT cells were plated at 15,000 cells/well and treated with DMSO (control), a control
  • oligonucleotide Compound No. 141923
  • an antisense oligonucleotide Compound No. 353382 or 399462
  • free uptake for 24 hours 0.01 mM, 0.1 mM, 1 mM, 2.5 mM, or 5 mM.
  • Cell treatment MHT cells were plated at 20,000 cells/well and treated with DMSO (control) or AZD8055 for 24 hours. Modified oligonucleotides described above were then added by free uptake for 40 minutes, 2 hours, or 4 hours, then the cells were incubated with fresh media (containing no oligonucleotide or autophagy modulator) for 2 hours prior to cell harvest.
  • RT-qPCR analysis was performed to detect RNA levels of Malat-l using primer probe set 15877 (described in Example 1). Results (shown in Table 23) were normalized to cyclophilin A levels.
  • Results shown in Table 23
  • Malat-l RNA levels after treatment with an antisense oligonucleotide and an autophagy modulator were normalized to Malat-l RNA levels in cells treated with control Compound No . 141923.
  • Example 8 Confocal imaging of autophagic vesicles
  • the Premo autophagy tandem sensor RFP-GFP-LC3B (ThermoFisher, P36239) was used to visualize autophagy in MHT cells.
  • Cells were treated with the Premo sensor and either DMSO or AZD8055 for 24 hours prior to the addition of modified antisense oligonucleotide, Compound No. 851810, at 0.1 mM or 2 pM by free uptake for 40 minutes, 2 hours, or 4 hours.
  • Confocal images were analyzed by counting the total number of autophagic vesicles per cell and the number of autophagic vesicles that colocalized with antisense oligonucleotide. Results represent the average of 8-12 images. Results are shown in Table 24.
  • Compound No. 851810 comprises a 5-10-5 MOE gapmer of the sequence
  • CTGCTAGCCTCTGGATTTGA SEQ ID NO: 30
  • AlexaFluor647 conjugated to the 5’-end of the oligonucleotide Every intemucleoside linkage of Compound No. 851810 is a phosphorothioate
  • MHT cells were treated with the Premo sensor and either DMSO or AZD8055 for 24 hours prior to the addition of 2 mM Compound No. 851810 by free uptake for 40 minutes, 2 hours, or 4 hours.
  • the media was then replaced with fresh media containing LysoTracker (ThermoFisher) and cells were imaged 2 hours later. Confocal images were analyzed by counting the total number of lysosomes per cell and the number of lysosomes that colocalized with antisense oligonucleotide. Results represent the average of 6-10 images and are presented in Table 25.
  • COPB2 regulates the process of autophagosomes fusing with lysosomes. Inhibiting COPB2 can result in abortive autophagy and an increase in autophagosomes.
  • MHT cells were plated at 10,000 cells/well and transfected with control siRNA or siRNA targeted to COPB2 using Lipofectamine RNAiMAX for 48 hours. An autophagy modulator or DMSO was then added for 24 hours. Fourteen hours after the addition of an autophagy modulator, a modified antisense or modified control oligonucleotide was added at 5 mM by free uptake for ten hours.
  • RT-qPCR analysis was performed to detect RNA levels of Malat-l as described in Example 1 above.
  • Malat-l RNA levels after treatment with siRNA, an antisense oligonucleotide, and an autophagy modulator were normalized to Malat-l RNA levels in cells treated with the same siRNA, control Compound No. 141923, and the same autophagy modulator.
  • Example 11 Confocal imaging of lysosomes and autophagic vesicles
  • the Premo autophagy tandem sensor RFP-GFP-LC3B was used to visualize autophagy in MHT cells.
  • LysoTracker was used to visualize lysosomes. MHT cells were treated with the Premo sensor and
  • LysoTracker for 2 hours then either DMSO or an autophagy modulator for 14 hours.
  • Compound No. 851810 was then added at 2 mM by free uptake for 10 hours prior to cell imaging. Confocal images were analyzed by counting the total number of autophagic vesicles per cell and lysosomes per cell as well as the number of autophagic vesicles and lysosomes that colocalized with the antisense oligonucleotide of Compound No. 851810. Results represent the average of 4-6 images. Results are presented in Tables 27 and 28.
  • EE CellLight early endosome marker was used to visualize early endosomes in MHT cells.
  • MHT cells were treated with CellLight EE and either DMSO or 500 nM AZD8055 for 14 hours prior to the addition of Compound No. 851810 at 2 mM by free uptake for 10 hours.
  • Confocal images were then obtained and analyzed by counting the total number of early endosomes per cell and the number of early endosomes that colocalized with the antisense oligonucleotide of Compound No. 851810. Results represent the average of 7-8 images. Results are presented in Table 29. Table 29: Colocalization of antisense oligonucleotide with autophagic vesicles
  • Example 13 Effects of modulating autophagy on modified oligonucleotide activity in vivo - AZD8055 treatment
  • mice were administered 5 mg/kg control oligonucleotide 141923, or 5 mg/kg antisense oligonucleotide 399462.
  • mice were administered 1% DMSO or 5 mg/kg AZD8055 in 1% DMSO at three time points over 24 hours: at the time of administration of the modified oligonucleotide, and 12 and 22 hours after administration of the modified oligonucleotide.
  • a group of control mice was administered PBS at time 0 and 1% DMSO at 0, 12, and 22 hours. Mice were sacrificed at 24 hours after administration of the modified oligonucleotide and tissues were collected for analysis.
  • Ribosomal Protein (Ser240/244) (2215S), and S6 Ribosomal Protein (2217S) antibodies were obtained from Cell Signaling Technology.
  • the antibody against LC3B (ab48394) was purchased from Abeam.
  • the antibody against alpha-tubulin (11224-1 -AP) was purchased from Proteintech Group.
  • Antibody against P62 (GP62-C) was purchased from Progen.
  • Anti-rabbit (7074S) secondary antibody conjugated to HRP was from Cell Signaling Technology.
  • IRDye® 800CW Donkey anti-Guinea Pig IgG (925-32411) secondary antibody was purchased from LI-COR, Inc. Results shown in Table 30 confirm that autophagy was activated in liver.
  • Atg7 mRNA levels were measured by RT-qPCR analysis as described in Example 1 above. Atg7 mRNA levels are a marker of the induction of autophagy in treated mouse livers. Results are presented as percent change of RNA, relative to PBS control treated with DMSO, normalized to mouse cyclophilin A measured using mouse primer probe set Cyclophilin-A (forward sequence
  • TTCTGTAGCTCAGGAGAGCACCCCTCCACCCCATTTGCTCGCAGTATCCT designated herein as SEQ ID NO: 40.
  • Results presented in Table 31 show that activation of autophagy in vivo significantly enhanced ASO activity as demonstrated by greater Malatl RNA reduction in the livers of mice treated with AZD8055.
  • Example 14 Effect of modulating autophagy by fasting on antisense activity in vivo
  • mice Groups of 4 8-week-old male C57bl/6J mice were administered 5 mg/kg control oligonucleotide 141923 or 5 mg/kg antisense oligonucleotide 399462. Following administration of modified oligonucleotide, groups of mice were either allowed free access to chow (control) or fasted overnight (16 hr fast). Mice were sacrificed at 16 hours after administration of the modified oligonucleotide and tissues were collected for analysis.
  • the antibody against alpha-tubulin (11224-1 -AP) was purchased from Proteintech Group.
  • Antibody against P62 (GP62-C) was purchased from Progen.
  • Anti-rabbit (7074S) secondary antibody conjugated to HRP was from Cell Signaling Technology.
  • IRDye® 800CW Donkey anti-Guinea Pig IgG (925-32411) secondary antibody was purchased from LI-COR, Inc. Table 32: Autophagy markers in vivo in fasted animals
  • Atg7 mRNA levels are a marker of the induction of autophagy in treated mouse livers.
  • Results are presented in Table 33 relative to mice treated with PBS and allowed normal access to chow. These results demonstrate that induction of autophagy with rapamycin leads to a significant enhancement of modified oligonucleotide acitivity in vivo.
  • Example 15 Effects of modulating antisense activity in vivo with autophagy activator rapamycin
  • mice Groups of 4 8-week-old male C57bl/6J mice were administered 5 mg/kg control oligonucleotide 141923 or 5 mg/kg antisense oligonucleotide 399462. Following administration of modified oligonucleotide, mice were administered 1% DMSO or 5 mg/kg rapamycin in 1% DMSO to activate autophagy at three time points over 24 hours: at the time of administration of the modified oligonucleotide, and 12 and 22 hours after administration of the modified oligonucleotide. A group of control mice was administered PBS at time 0 and 1% DMSO at 0, 12, and 22 hours. Mice were sacrificed at 24 hours after administration of the modified oligonucleotide and tissues were collected for analysis.
  • the antibody against alpha-tubulin (11224-1 -AP) was purchased from Proteintech Group.
  • Antibody against P62 (GP62-C) was purchased from Progen.
  • Anti-rabbit (7074S) secondary antibody conjugated to HRP was from Cell Signaling Technology.
  • IRDye® 800CW Donkey anti-Guinea Pig IgG (925-32411) secondary antibody was purchased from LI-COR, Inc. Results presented in Table 34 demonstrate that autophagy was activated in liver.
  • Atg7 mRNA levels were measured by RT-qPCR analysis as described in Example 1 above. Atg7 mRNA levels are a marker of the induction of autophagy in treated mouse livers. Results are presented relative to mice treated with PBS and 1% DMSO. Results presented in Table 35 demonstrate that autophagy activation enhances antisense activity in vivo.
  • Example 16 Effect of modulating autophagy by fasting on modified oligonucleotide activity in vivo - ketogenic diet
  • mice were subjected to a 5 -week ketogenic diet which has been shown to inhibit mTORCl activity and activate autophagy in mice.
  • Mice were treated with either PBS, 5 mg/kg of Compound No. 549148, added as a control modified oligonucleotide, or 5mg/kg of Compound No. 556089 that targets mouse MALAT1 (total of 1 administration of modified oligonucleotide) at the end of the 5 -weeks on the ketogenic diet.
  • Mice were sacrificed 16 hours post modified oligonucleotide administration and liver tissue was collected to examine the effects of autophagy induction on modified oligonucleotide activity.
  • Ribosomal Protein (Ser240/244) (2215S), and S6 Ribosomal Protein (2217S) antibodies were obtained from Cell Signaling Technology.
  • the antibody against LC3B (ab48394) was purchased from Abeam.
  • the antibody against alpha-tubulin (11224-1 -AP) was purchased from Proteintech Group.
  • Antibody against P62 (GP62-C) was purchased from Progen.
  • Anti-rabbit (7074S) secondary antibody conjugated to HRP was from Cell Signaling Technology.
  • IRDye® 800CW Donkey anti-Guinea Pig IgG (925-32411) secondary antibody was purchased from LI-COR, Inc. Results presented in Table 36 demonstrate that autophagy was activated in liver.
  • Primer probe set 15877 was used to measure mouse MALAT1 RNA levels. Results are presented as percent change of RNA, relative to PBS control, normalized to mouse cyclophilin A measured using mouse primer probe set Cyclophilin-A.
  • Results presented in Table 37 show that diet induced activation of autophagy in vivo significantly enhanced antisense activity as demonstrated by greater Malatl RNA reduction in the livers of mice on a ketogenic diet.
  • Example 17 Time course of antisense activity in vivo with ketogenic diet induced autophagy
  • mice were subjected to a 5 -week ketogenic diet which has been shown to inhibit mTORCl activity and activate autophagy in mice.
  • Mice were treated with either PBS, 5 mg/kg of Compound No. 549148, added as a control modified oligonucleotide, or 5mg/kg of Compound No. 556089 that targets mouse MALAT1 (total of 1 administration of modified oligonucleotide) at the end of the 5-weeks on the ketogenic diet.
  • Mice were sacrificed 24, 48 and 72 hours post modified oligonucleotide administration and liver tissue was collected to examine the effects of autophagy induction on modified oligonucleotide activity.
  • Primer probe set 15877 was used to measure mouse MALAT1 RNA levels. Results are presented as percent change of RNA, relative to PBS control, normalized to mouse cyclophilin A measured using mouse primer probe set Cyclophilin-A.
  • Example 18 Effect of modulation of autophagy on antisense activity in human fibroblasts
  • Human fibroblasts were plated at 8000 cells/well and treated with DMSO (control) or an autophagy modulator (AZD8055) for 24 hours, at the concentrations indicated in Example 1.
  • DMSO control
  • AZD8055 autophagy modulator
  • Compound No. 556089 was added at 10mM by free uptake for ten hours, as indicated in the tables below.
  • RT-qPCR analysis was performed to detect RNA levels of Malat-l using primer probe set RTS2736. Results were normalized to cyclophilin A levels.
  • Example 19 Effect of modulation of autophagy on antisense activity in primary mouse hepatocytes
  • Compound No.841226 (a 5-10-5 MOE gapmer that targets mouse MALAT1 and has the sequence (from 5’ to 3’): CCAGGCTGGTTATGACTCAG, with a Cy3 (IDT 5Cy3 fluorophore) conjugate on the 5’ end of the sequence and a THA-C6-GalNAc hydroxyproline PO conjugate at the 3’ end of the sequence, designated herein as SEQ ID No. 41.
  • Every intemucleoside linkage of Compound No. 841226 is a phosphorothioate intemucleoside linkage. All of the cytosines had a methyl at the 5 position.
  • RT-qPCR analysis was performed to detect RNA levels of Malat-l using primer probe set previously described in Example 1. Results were normalized to cyclophilin A levels. Induction of autophagy significantly enhanced activity of modified oligonucleotides (both unconjugated and conjugated MOE gapmers) targeting MALAT1 in mouse primary hepatocytes, as evidenced by the examples presented in Tables 40 and 41.
  • Example 20 Effect of modulation of autophagy on antisense activity in mouse embryonic fibroblasts
  • Mouse embryonic fibroblasts were plated at 8000 cells/well and treated with DMSO (control) or an autophagy modulator (AZD8055) for 24 hours, at the concentrations indicated in Example 1.
  • Compound No. 353382 was added at 3000nM, lOOOnM and 500nM by free uptake for ten hours, as indicated in the tables below.
  • mouse embryonic fibroblasts (MEFs) were plated at 8000 cells/well and treated with DMSO (control) or an autophagy modulator (AZD8055) for 24 hours, at the concentrations indicated in Example 1.
  • DMSO control
  • autophagy modulator ALD8055
  • Compound No. 399462 was added at lOOOnM, 500nM and 250nM by free uptake for ten hours, as indicated in the tables below.
  • RT-qPCR analysis was performed to detect mRNA levels of SRB1 using primer probe set 15299, and to detect MALAT1 levels using primer probe set 15877. Results were normalized to cyclophilin A levels and are presented as relative to untreated control (%UTC). Induction of autophagy significantly enhanced activity of modified oligonucleotides against multiple targets in mouse embryonic fibroblasts, as evidenced by the results presented in Tables 42 and 43.
  • Example 21 Effect of modulation of autophagy on antisense activity in human lung epithelial cells
  • H460 cells were plated at 7500 cells/well and treated with DMSO (control) or an autophagy modulator (AZD8055) for 24 hours, at the concentrations indicated in Example 1. Fourteen hours after the addition of an autophagy modulator, Compound No. 395254 was added at lOOOnM by free uptake for ten hours.
  • RT-qPCR analysis was performed to detect mRNA levels of MALAT1 using primer probe set RTS2736. Results were normalized to cyclophilin A levels and are presented as relative to untreated control (%UTC).
  • Example 22 Effect of modulation of autophagy on antisense activity in human brain neuroglioma cells
  • H4 cells (a cell line derived from a human brain neuroglioma) were plated at 7500 cells/well and treated with DMSO (control) or an autophagy modulator (AZD8055) for 24 hours, at the concentrations indicated in Example 1. Fourteen hours after the addition of an autophagy modulator, Compound No. 395254 was added at 5000nM by free uptake for ten hours.
  • DMSO control
  • AZD8055 autophagy modulator
  • RT-qPCR analysis was performed to detect mRNA levels of MALAT1 using primer probe set RTS2736. Results were normalized to cyclophilin A levels and are presented as relative to untreated control (%UTC).
  • Example 23 Effect of modulation of autophagy on antisense activity in vitro in HT1080 cells
  • HT1080 cells were plated at 7500 cells/well and treated with DMSO (control) or an autophagy modulator (AZD8055) for 24 hours, at the concentrations indicated in Example 1.
  • DMSO control
  • autophagy modulator ALD8055
  • Table 46 All the compounds described in Table 46 have uniform phosphorothioate intemucleoside linkages. All of the cytosines had a methyl at the 5 position. All compounds were added to cells at 5000nM.
  • RT-qPCR analysis was performed to detect RNA levels of STAT3 using primer probe set RTS96 (forward sequence AATGGCTAAGTGAAGATGACAATCAT, designated herein as SEQ ID NO: 44; reverse sequence TGCACATATCATTACACCAGTTCGT, designated herein as SEQ ID NO: 45; probe sequence TTGCAGCAATTCACTGTAAAGCTGGAAAGG, designated herein as SEQ ID NO: 46).
  • PTEN RNA levels were detected using Thermofisher primer probe set Hs00374280_ml .
  • c-myc RNA levels were detected using Thermofisher primer-probe set Hs00l53408_ml . Results were normalized to cyclophilin A levels (measured using HTS3936) and are presented as relative to untreated control (%UTC).

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Abstract

L'invention concerne des procédés d'augmentation de l'activité oligonucléotide antisens dans une cellule par modulation de l'autophagie de la cellule. Dans certains modes de réalisation, un composé comprenant un oligonucléotide antisens est co-administré à un sujet avec un modulateur d'autophagie.
EP19782173.9A 2018-04-06 2019-04-04 Procédés de modulation de l'activité antisens Withdrawn EP3775215A1 (fr)

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